AU3521499A - Medicament for preventing or treating papilloma virus-specific tumors - Google Patents

Description

WO 99/48518 PCT/EP99/01996 Medicament for the avoidance or treatment of papillomavirus-specific tumour The present invention relates to a medicament for the 5 avoidance or treatment of human papillomaviruses (HPV) specific tumour comprising at least one fusion protein from at least one Li protein of one or more papillomaviruses and at least one E protein of one or more papillomaviruses and, if appropriate, suitable 10 additives and/or excipients, the fusion protein containing no papillomavirus-unspecific epitopes. Papillomaviruses, also called wart viruses, are double stranded DNA viruses having a genome size of 15 approximately 8000 base pairs and an icosahedral-like capsid having a diameter of about 55 nm. To date, more than 100 different human papillomavirus types are known, of which some, e.g. HPV-16, HPV-18, HPV-31, HPV-33, HPV-39, HPV-45, HPV-52 or HPV-58, can cause 20 malignant tumours and others, e.g. HPV-6, HPV-11 or HPV-42, can cause benign tumours. Electron-microscopic analysis of BPV-1 and HPV-1 shows that the viruses are constructed from 72 pentameric 25 capsomers, which in turn consist of five Li molecules (Baker, T. et al. (1991) Biophys. J., 60, 1445). The genome of the papillomaviruses can be subdivided into three areas: the first area concerns a non-coding 30 region which contains regulation elements for the transcription and replication of the virus. The second region, the so-called E (early) region, contains various protein-coding sections El-E7, of which, for example, the E6 protein and the E7 protein is [sic] 35 responsible for the transformation of epithelial cells and the El protein controls the DNA copy number. The E6 region and E7 region are so-called oncogenes, which are also expressed in malignantly degenerate cells. The third region, also called the L (late) region, contains WO 99/48518 - 2 - PCT/EP99/01996 two protein-coding sections Li and L2, which code for structural components of the virus capsid. The Li protein is present to over 90% in the viral capsid, the ratio of Ll:L2 in general being 30:1. 5 HPV-6 and HPV-11 have been held responsible, inter alia, for genital warts; some papillomavirus types such as HPV-16, HPV-18, HPV-31, HPV-33, HPV-39, HPV-45, HPV-52 and HPV-58 are associated with malignant tumours 10 of the anogenital tract. In over 50% of the cases, HPV-16 is connected with cervical cancer (carcinoma of the cervix). HPV-16 is therefore the main risk factor for the formation of cervical neoplasias. In addition, the immune system plays an important role in the 15 progress of the disease. Thus cellular immune responses and in particular antigen-specific T lymphocytes are supposedly important for the defence mechanism. It has furthermore been found that in high-grade cervical intraepithelial neoplasias (CIN II/III) and cervical 20 tumour the E7 gene is expressed constitutively in all layers of the infected epithelium. The E7 protein is therefore considered as a potential tumour antigen and as a target molecule for activated T cells (see, for example, WO 93/20844). The E7-induced cellular immune 25 response in the patient, however, is apparently not strong enough to influence the course of the disease. The immune response can possibly be amplified by suitable vaccines. 30 It has now been possible to show that the expression of the Li gene or the coexpression of the Li and L2 gene forms virus-like particles (VLPs). It was possible to use the VLPs for the formation of neutralizing antibodies in various animal systems. The formation of 35 virus-neutralizing antibodies, however, is of relatively low clinical importance if the virus c ~ infection has already taken place, since for the "'>elimination of virus-infected cells a virus-specific WO 99/48518 - 3 - PCT/EP99/01996 cytotoxic T-cell (CTL) response appears to be necessary. So-called chimeric papillomavirus-like particles (CVLPs) were therefore developed which consist of a chimeric Ll-E7 protein (MUller, M. et al. 5 (1997) Virology, 234, 93): some CVLPs induce an E7-specific CTL response in mice, although experiments failed to induce antibodies by immunization of mice with CVLPs against E7 (Miller, M. et al. (1997), supra). In addition, neutralizing antibodies of HPV 10 associated disorders in patients appear to limit the immune response to administered Ll protein (MUller, M. et al. (1997), supra). CVLPs, however, are still of interest for the development of a vaccine, as the E7 proteins of tumour cells presented via MHC molecules 15 of class I would represent target molecules of CTLs. Peng et al. (1998) Virology, 240, 147 now describe CVLPs consisting of C-terminally truncated Ll of the bovine papillomavirus (BPV) and HPV-16E7 4 9

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5 7 , which 20 induce E7-specific cytotoxic T cells after inoculation of C57B1/6 mice and protect against the growth of E7-expressing tumours. Greenstone et al. (1998) Proc. Natl. Acad. Sci. USA, 95, 1800 describe CVLPs consisting of HPV-16L1 plus HPV-16L2 fused to the full 25 length HPV-16E7 protein, which protect against the growth of epithelial E7-expressing tumour cells after immunization of C57B1/6 mice, cytotoxic T cells, however, not being detected and thus the induction of the immune response appearing to be less efficient. 30 VLPs and CVLPs are in general prepared by means of genetic engineering by expression of the corresponding genes coding for one or more L proteins or L and E proteins in suitable expression systems. The 35 corresponding genes are described, for example, in Kirnbaum, R. et al. (1994) J. Virol., 67, 6929-6936 or obtainable via the EMBL databank. The accession numbers C-O4.4 -7 ~CI WO 99/48518 - 4 - PCT/EP99/01996 are, for example, for HPVl8: PAPHPVl8; for HPV31: PAPPPH31; for HPV33: PAPPPH33 or for HPV58: PAPPPH58. Suitable expression systems are, for example, yeasts 5 modified by genetic engineering, e.g. Saccharomyces (cerevisiae), Pichia (pastoris), Kluyvermyces (lactis), Schizosaccharomyces (pombe) or Hansenula (polymorpha) (Carter, J. J. et al. (1991), Virology, 182, 513), insect cells, such as, for example, Trichoplusia ni 10 High Five (see, for example, MUller et al. (1997), supra) or prokaryotic cells (see, for example, WO 96/11272). In the case of the production of the particles in prokaryotic cells, these are in general deposited in the cell and form so-called inclusion 15 bodies, which then have to be renatured and brought into solution. For use of the particles or capsids produced by genetic engineering or their precursors, the so-called capsomers, further purification steps are necessary after expression. 20 A crucial disadvantage of the active compounds against HPV described in the literature is, however, that on the one hand they exhibit only a slight action and on the other hand that it has not been possible to date to 25 show any effective immunotherapy of papillomavirus specific tumour. It was therefore the object of the present invention to make available a medicament with which human 30 papillomavirus-specific tumour can be avoided or treated effectively, which can be produced simply and which appears to be suitable for licensing as a medicament. 35 It has now surprisingly been found that the medicament according to the invention is effective against HPV R" specific tumour. 4, ro- WO 99/48518 - 5 - PCT/EP99/01996 One subject of the present invention is therefore a medicament for the avoidance or treatment of human papillomavirus (HPV) -specific tumour comprising at least one fusion protein from at least one Li protein 5 of one or more papillomaviruses and at least one E protein of one or more papillomaviruses and, if appropriate, suitable additives and/or excipients, the fusion protein comprising no papillomavirus-unspecific epitopes. 10 Papillomavirus-unspecific epitopes within the meaning of the present invention are understood in general as meaning epitopes in the fusion protein which are caused by a foreign protein fraction, by post-translational 15 modifications or by misfolding of papillomavirus specific proteins. The papillomavirus-unspecific epitopes are, for example, a reason for the fact that although 20 neutralizing antibodies or CTL immune responses are induced, the papillomavirus-specific tumour cannot be effectively avoided or controlled, as the immunological action is weakened by unspecific antibodies or CTLs or immunological side effects interfere with the action of 25 the actual active compound. The medicament according to the invention is preferably effective for the avoidance or treatment of benign or malignant tumour, in particular of carcinoma of the 30 larynx, cervix, penis, vulva or anus, including their preliminary stages, such as, for example, high-grade CIN (cervical intraepithelia neoplasia). In a further preferred embodiment, the medicament 35 according to the invention contains no adjuvant, i.e. no substance which amplifies the immunity of the papillomavirus-specific protein, as the presence of an L protein in particular, especially of L1, already WO 99/48518 - 6 - PCT/EP99/01996 adequately amplifies the immunity. This property is particularly advantageous in the licensing as a medicament or diagnostic, as the only immunostimulating materials licensed by the licensing authorities at 5 present are aluminium salts. Moreover, undesired side effects are avoided by the omission of adjuvants and/or other excipients and additives. As already mentioned above, a further significant 10 problem in the use of capsids and capsomers as medicaments is their poor solubility. Thus capsids or capsomers of HPV-16, for example, tend to aggregate, whereby the solubility is significantly reduced. The solubility of the capsids or capsomers, which in some 15 cases is low, leads not only to a loss of yield, but also to complicated use as a medicament. In a further preferred embodiment, the medicament according to the invention therefore contains as a 20 suitable additive or excipient about 0.3 to about 4 M, preferably about 0.4 to about 3 M, in particular about 0.5 to 2 M, especially about 1 to about 2 M, of a salt having a pH of about 7.3 to about 7.45, preferably about 7.4. 25 The advantage of this salt solution is that the fusion protein remains in solution or is present finely divided as a suspension, i.e., in general more than about 90%, especially more than about 95%, of the 30 fusion protein remains in solution and also does not deposit for a period of time of at least about 12 hours. The fusion protein is also not significantly sedimentable by centrifugation at a maximum of 5000 g. 35 The salt is in general an alkali metal or alkaline earth metal salt, preferably a halide or phosphate, in particular an alkali metal halide, especially NaCl WO 99/48518 - 7 - PCT/EP99/01996 and/or KCl. Use of NaCl is particularly preferred for the production of a pharmaceutical formulation. The pH of the medicament is in general adjusted using a 5 suitable organic or inorganic buffer, such as, for example, preferably using a phosphate buffer, tris buffer (tris (hydroxymethyl) aminomethane), HEPES buffer ([4- (2-hydroxyethyl)piperazino] ethanesulphonic acid) or MOPS buffer ( 3 -morpholino-l-propanesulphonic acid). The 10 choice of the respective buffer in general depends on the desired buffer molarity. Phosphate buffer is suitable, for example, for injection and infusion solutions. 15 Suitable further additives and/or excipients which serve, for example, for the further stabilization of the papillomavirus-specific protein in the medicament according to the invention are, for example, detergents, such as, for example, Triton X-100 or 20 sodium deoxycholate, but also polyols, such as, for example, polyethylene glycol or glycerol, sugars, such as, for example, sucrose or glucose, zwitterionic compounds, such as, for example, amino acids such as glycine or in particular taurine or betaine and/or a 25 protein, such as, for example, bovine or human serum albumin. Detergents, polyols and/or zwitterionic compounds are preferred. Other additives and/or excipients are protease inhibitors, such as, for example, aprotinin, E-aminocaproic acid or pepstatin A. 30 Those additives are preferred which induce no immunological side effects. The terms Ll/L2 protein and E protein are understood within the meaning of the present invention as meaning 35 both the full-length proteins and their mutants, such as, for example, deletion mutants. S

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1 WO 99/48518 - 8 - PCT/EP99/01996 In a further preferred embodiment, the fusion protein according to the invention contains a deleted L protein, preferably a deleted Ll and, if appropriate, L2 protein. The deletion has the advantage that 5 particularly active different proteins, for example papillomavirus-specific E protein sequences, can be inserted into the deleted area, whereby the application area of the composition according to the invention can be widened. An L protein having a C terminal deletion 10 and in particular a C-terminally deleted Ll protein is particularly preferred. The C-terminal deletion has the advantage that the efficiency of the formation of virus-like particles can be increased, since the nuclear location signal located at the C terminus is 15 deleted. The C-terminal deletion is therefore preferably up to about 35 amino acids, in particular about 25 to about 35 amino acids, especially about 32 to 34 amino acids. For example, a C-terminal deletion of the HPV-16L1 protein 32 amino acids long is adequate 20 to be able to increase the formation of virus-like particles by at least about 10-fold. In a further preferred embodiment, the E protein is also deleted, especially the E6 and/or E7 protein. It 25 is particularly preferred if the C-terminal part of the E protein is deleted, preferably the C-terminal part of the E7 protein, as these constructs can preferably form capsomers and/or capsids in combination with deleted L protein. Deletions of up to 55 amino acids are 30 particularly preferred, preferably about 5 to about 55 amino acids, in particular about 32 to about 43 amino acids. A particularly preferred construct is, for example, E7 35 having the N-terminal amino acids 1 to about 60, as this construct contains a mouse epitope for the activation of cytotoxic T lymphocytes, which is located in the area of the amino acids 49-57. Another preferred

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WO 99/48518 - 9 - PCT/EP99/01996 construct is E7 having the N-terminal amino acids 1 to about 55, which preferably forms capsomers and capsids in combination with deleted L protein, as this construct does not contain E7-specific sequences in the 5 area of the amino acids 56-70, which can interfere with the formation of capsids. An Ll protein of HPV-16 C-terminally deleted by 32 amino acids and which is linked to an E7 protein of HPV-16 having the amino acids 1-55 or 1-60 is particularly preferred. These 10 constructs not only induce neutralizing antibodies or a specific CTL response, but on the one hand prevents [sic] the formation of tumours and on the other hand cause regression of already existing tumours in animal experiments. E7 having the amino acids 1-60 especially 15 exhibits a marked prophylactic and therapeutic action in tumours. A particularly preferred embodiment of the present invention is therefore an L1AE7 1 _x fusion protein, preferably in the form of a CVLPs, in particular of HPVl6, x being an integer from 55 up to 20 and including 60, and in particular an L1AE7 1

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5 5 [sic] or L1AE7 1 -6 0 [sic] fusion protein. The present invention therefore also relates to the use of the constructs according to the invention for the 25 production of a medicament, on the one hand for the connection [sic] of HPV-specific tumours and on the other hand for the regression of already existing HPV specific tumours. 30 For the production of a medicament which is active both prophylactically and therapeutically, it is preferred if the described papillomavirus-specific fusion protein is present in the form of a capsid and/or capsomer, since the immune reaction can additionally be markedly 35 increased by the capsids and/or capsomers and in particular by the fraction of L protein. Preferred fusion proteins which are suitable for. capsid and/or WO 99/48518 - 10 - PCT/EP99/01996 capsomer formation are therefore, for example, fusion proteins from deleted Li and E7, E6 and/or El. Capsids within the meaning of the present invention are 5 viral or virus-like structures in a generally icosahedral form, which in general are constructed of 72 capsomers. Capsomers within the meaning of the present invention 10 are assembled proteins comprising at least one papillomavirus structural protein, preferably Li or deletions of Li. For example, 5 fusion proteins according to the invention can be assembled to give a capsomer which in turn can be assembled to give a 15 capsid. For the production of a human medicament, proteins or peptides of the human papillomavirus (HPV) and preferably of HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, 20 HPV-33, HPV-35, HPV-39, HPV-45, HPV-52 and/or HPV-58, in particular HPV-16, HPV-18, HPV-31 and/or HPV-45 are suitable for the constructs described. Especially for the production of a combination vaccine, it is advantageous to combine proteins or peptides from 25 various HPV types, for example a combination of HPV-16 and HPV-18 or HPV-18, HPV-31, HPV-45 and HPV-58 in the case of, for example, carcinoma of the cervix or HPV-6 and HPV-11 in the case of, for example, condylomas. 30 A further subject of the present invention is a process for the production of a medicament according to the invention, in which a suitable cell comprising a suitable expression vector which codes for the fusion protein mentioned is cultured under suitable 35 conditions, the expression product is isolated and, if appropriate, suitable additives and/or excipients are added.

WO 99/48518 - 11 - PCT/EP99/01996 The expression vectors can be, for example, prokaryotic or eukaryotic expression vectors. Examples of prokaryotic expression vectors are, for expression in E. coli, e.g. the vectors pGEM or pUC derivatives (see, 5 for example, WO 96/11272). Examples of eukaryotic expression vectors are, for expression in Saccharomyces cerevisiae, e.g. the vectors p426Met25 or p426GALl (Mumberg et al. (1994) Nucl. Acids Res., 22, 5767-5768, Carter, J. J. et al. (1991) supra) and, for expression 10 in insect cells, e.g. Baculovirus vectors, in particular the Autographa Californica virus, such as disclosed in EP-B1-0 127 839 or EP-B1-0 549 721 (see, for example, also WO 94/20137), and, for expression in mammalian cells, e.g. the vectors Rc/CMV and Rc/RSV or 15 SV40 vectors which are all generally obtainable. However, commercially obtainable Baculovirus expression systems are also suitable, such as, for example, the Baculo GoldTM transfection kit from Pharmingen or the Bac-to-BacTM Baculovirus expression system from 20 Gibco BRL. Further suitable expression systems are recombinant vaccinia viruses (see, for example WO 93/02184). In general, the expression vectors also contain 25 promoters suitable for the respective host cell, such as, for example, the trp promoter for expression in E. coli (see, for example, EP-B1-0 154 133), the ADH2 promoter for expression in yeasts (Russel et al. (1983), J. Biol. Chem. 258, 2674-2682), the Baculovirus 30 polyhedrin promoter for expression in insect cells (see, for example EP-B1-0 127 839 or U.S. 5,004,687) or the early SV40 promoter or LTR promoters, e.g. of MMTV (mouse mammary tumour virus; Lee et al. (1981) Nature 214, 228-232). 35 Suitable host cells are, for example, the E. coli strains DH5, HB101 or BL21, the yeast strains Saccharomyces, Pichia, Kluyvermyces, Schizosaccharomyces

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WO 99/48518 - 12 - PCT/EP99/01996 or Hansenula (Carter, J. J. et al. (1991), Virology, 182, 513), the insect cell line Lepidopteran, e.g. from Spodoptera frugiperda, Trichoplusia ni, Rachiplusia ou or Galleria Mellonela or the animal cells COS, C127, 5 Vero, 293 and HeLa, which are all generally obtainable (see, for example, WO 94/00152). The coding nucleic acids for the individual papillomavirus-specific proteins can be isolated and 10 cloned, for example, from a gene bank by means of a PCR (polymerase chain reaction) amplification. For example, the genome of BPV-1 is generally obtainable under the GenBank Accession No. X02346 or HPV-16 under the GenBank Accession No. K02718. An HPV-16 Ll sequence is 15 also disclosed, for example, in WO 94/05792. The sequence of the 98 amino acid-long HPV16 E7 protein is described, for example, in Seedorf et al. (1985) Virology, 145, 181-185. Another method of obtaining the desired nucleic acids is to isolate the papillomavirus 20 specific genes directly from warts or tumours by means of PCR. Suitable primers for the E6 and E7 genes from HPV-16 and HPV-18 are disclosed, for example, in WO 93/21958. Further references for the desired nucleic acids are, for example, Kirnbaum, R. et al. (1994), 25 supra or the clones deposited in the EMBL databank already mentioned above. In a further preferred embodiment, the expression vector is constructed such that the expressed fusion 30 protein is extended by no further amino acids caused by the vector. This is achieved, for example, by removing undesired nucleotides which code for additional amino acids by mutagenesis in a PCR reaction by means of suitable primer oligonucleotides (Ho et a. [sic] (1989) 35 Gene, 77, 51-59). In this way, a fusion protein is obtained which is free of additional amino acids and 7r >thus free of possible additional foreign epitopes which Fan cause immunological side reactions.

WO 99/48518 - 13 - PCT/EP99/01996 After the expression of the described fusion protein, it is preferred to purify this further or to renature it. Examples of chromatographic purification processes 5 are found in Hjorth, R. & Moreno-Lopez, L. (1982) J. Virol. Meth. 5, 151; Nakai, Y. et al. (1987) J. Gen. Virol., 68, 1891 [sic]; Hofmann, K. J. et al. (1995) Virology, 209, 506; Rose, R. C. et al. (1993) J. Virol., 67, 1936, Sasagawa, T. et al. (1995) 10 Virology, 206, 126 or WO 95/31532. In general, the medicament can be administered orally, parenterally, such as, for example, subcutaneously, intramuscularly or via the mucous membrane, in liquid 15 or suspended form, in the form of an elixir or as capsules, preferably as an injection or infusion solution. In the case of the formulations according to the invention, an adjuvant can be dispensed with, which is particularly advantageous. 20 A further subject of the present invention therefore relates to the use of the formulation according to the invention as an injection or infusion solution. 25 Injection solutions are in general used if only relatively small amounts of a solution or suspension, for example about 1 to about 20 ml, are to be administered to the body. Infusion solutions are in general used if a larger amount of a solution or 30 suspension, for example one or more litres, are to be administered. Since, in contrast to the infusion solution, only a few millilitres are administered in the case of injection solutions, small differences from the pH and from the osmotic pressure of the blood or 35 the tissue fluid in the injection do not make themselves noticeable or only make themselves \ST noticeable to an insignificant extent with respect to pain sensation. Dilution of the formulation according WO 99/48518 - 14 - PCT/EP99/01996 to the invention before use is therefore in general not necessary. In the case of the administration of relatively large amounts, however, the formulation according to the invention should be diluted briefly 5 before administration to such an extent that an at least approximately isotonic solution is obtained. An example of an isotonic solution is a 0.9% strength sodium chloride solution. In the case of infusion, the dilution can be carried out, for example, using sterile. 10 water while the administration can be carried out, for example, via a so-called bypass. The figures and the following examples are intended to illustrate the invention in greater detail without 15 restricting it. Description of the Figures Fig. 1 shows an E7-specific CTL response which were 20 [sic] produced after immunization with 1 gg of LlE7 1

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60 CVLPs. The isolated spleen cells of mice immunized with LlE7 1

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6 0 CVLPs (filled symbols) and HBS buffer (open symbols) were stimulated once in vitro with irradiated RMA-E7 cells and tested after five days in a standard 25 four-hour 51Cr release cytotoxicity test with the following target cells: RMA-E7 cells (squares), E7 4 9

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5 7 peptide-loaded RMA cells (triangular [sic]) and RMA cells (circles) . The results are expressed as specific lysis in %. 30 Fig. 2 shows the result of a titration test, carried out using an E7-specific CTL line which was obtained after vaccination of a C57BL/6 mouse with 20 gg of L1E7 1

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6 0 CVLPs and in vitro stimulation three times with 35 RMA-E7 transfectants. The peptide concentration was between 100 pg and 1 gg/ml. The target cells used were 51 Cr-labelled E7 49-57 peptide-loaded RMA cells. The ratio of cells to target cells was 30:1.

WO 99/48518 - 15 - PCT/EP99/01996 Fig. 3 shows the protection of C57BL/6 mice against the growth of TC-1 tumour cells. The mice (5 per group) were immunized s.c. either with 10 gg of L1E7 1 -6 0 CVLPs 5 (triangular [sic]), with 10 gg of L1AC VLPs (circles) or with HBS buffer (squares). Two weeks later, 6 X 104 TC-1 tumour cells per mouse were inoculated into the left flank of the mice s.c.. The mice were checked twice per week. 10 Fig. 4 shows the prevention of the growth of TC-1 tumour by. L1E7 1

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60 CVLPs. 6 x 104 TC-1 tumour cells per mouse were inoculated into the left flank of C57BL/6 mice (5 per group). Two weeks later, the mice 15 were immunized with an s.c. injection of 10 gg of L1E7 1 -6 0 CVLPs (triangles), 10 gg of L1ACVLPs (circles) or HBS buffer (squares). Examples 20 1. Preparation of chimeric genes coding for HPV16L1E7 fusion proteins The HPV-16L1 open reading frame (ORF) was excized from 25 the plasmid HPV-16-114/k-Ll/L2-pSynxtVI~ (Kirnbauer, R. et al. (1994) J. Virol. 67, 6929) using the restriction endonuclease BglII and cloned into the BamHI site in the vector pUC19 (New England Biolabs). 30 For the preparation of HPV-16L1AC, two primers were constructed which are complementary to HPV-16L1 ORF. The first primer has the sequence AAAGATATCTTGTAGTAAAAATTTGCGTCCTAAAGGAAAC and the second primer [lacuna] 35 AAAGATATCTAATCTACCTCTACAACTGCTAAACGCAAAAAACG Both primers encode an EcoRV restriction enzyme cleavage site 5'. In the primers lying downstream, a WO 99/48518 - 16 - PCT/EP99/01996 TAA translation stop codon follows the EcoRV site in order to delete the last 34 amino acids of the HPV16L1 ORF. The PCR reaction was carried out in order to amplify the entire Li ORF and the entire vector. The 5 linear product was cleaved with EcoRV, circularized with T4 DNA ligase and transformed [lacuna] E. coli DH5L cells. The clones were analysed for the presence of an EcoRV site. The construct pUCHPVl6L1AC obtained was used in order to clone the ORF of HPV16E7 1-50 into 10 the EcoRV site. For the cloning of the fragment, primers having a 5'EcoRV restriction enzyme cleavage site were used. The following primer pair was used: 15 AAAAGATATCATGCATGGAGATACACCTACATTGC and TTTTGATATCGGCTCTGTCCGGTTCTGCTTGTCC. The PCR products were cleaved with EcoRV and inserted 20 into the EcoRV site of the modified Li gene. For the elimination of the EcoRV sites, two PCR reactions were carried out in order to amplify two overlapping fragments of the clone pUC-HPV16LlACE7 25 1-50. The resulting DNA fragments overlapped in the position of the Ll/E7 boundary (Four Primer PCR, Ho, S. N. et al (1989) Gene 77, 51). However, the primers did not contain the two EcoRV restriction enzyme cleavage sites. Fragment 1 was prepared using 30 the primers P1 and P2 and fragment 2 using the primers P3 and P4. P1: GTTATGACATACATACATTCTATG (L1) P2: CCATGCATTC TGCTTGTAGTAAAAATTTGCGTCC (E7) P3: CTACAAGCA ;AATGCATGGAGATACACC (E7) P4:CATCTGAAGCTTAGTAATGGGCTCTGTCCGGTTCTG (E7) WO 99/48518 - 17 - PCT/EP99/01996 A tenth of the purified products was mixed and used as a matrix in the PCR reaction with the primers P1 and P4 exclusively. The resulting product was cleaved using EcoNI (Li) and HindIII (downstream of the stop codon on 5 the primer P4) and used in order to replace an EcoNI/HindIII fragment of the cloned HPV16L1 ORF. The resulting clone therefore differs from the clone HPV16L1ACE7 1-50 by the loss of the two internal EcoRV restriction enzyme cleavage sites and the corresponding 10 non-HPV amino acids Asp and Ile between the Ll ORF and E7 and downstream of E7. The first EcoRV site was replaced by the original Li amino acids in this position (AlaGly) . The second EcoRV site was replaced by a translation stop signal. This clone (HPV16L1AC*E7 15 1-52) additionally contains the first 52 amino acids of HPV16E7. Clone HPV16L1AC*E7 1-52 was used for the preparation of the clones HPV16LlAC*E7 1-55, 1-60 and 1-65 with the aid of the primer P1 in combination with P5, P6 and P7. 20 P5: CATCTGAAGCTTATCAATATTGTAATGGGCTCTGTCCG (E7 1-55) P6 CATCTGAAGCTTACTTGCAACAAAAGGTTACAATATTGT AATGGGCTCT GTCCG (E7 1-60) P7 CATCTGAAGCTTAAAGCGTAGAGTCACACTTGCAACAAAAGGTTACAA TATTGTAATGGGCTCTGTCCG (E7 1-65). HPV16L1AC*E7 1-70 was prepared using the clone HPV16L1AC*E7 1-65 and the primers P1 and P8. 25 P8: CATCTGAAGCTTATTGTACGCACAACCGAAGCGTAGAGTCACACTTG In all cases, EcoNI and HindIII were used in order to <> replace the corresponding fragments. The clones were 34 analysed by DNA sequencing.

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WO 99/48518 - 18 - PCT/EP99/01996 2. Preparation of recombinant Baculoviruses Spodoptera frugiperda (Sf9) cells were used as a 5 monolayer or in suspension culture in TNM-FH insect medium (Sigma, Deisenhofen) with 10% foetal calf serum and 2 mM glutamine. Recombinant baculoviruses HPV16L1ACE7 1-x were transfected by cotransfection of 10 ug of the recombinant plasmids and 2 gg of 10 linearized Baculo-Gold DNA (Pharmingen, San Diego, CA) into Sf9 cells. Recombinant viruses were purified according to the instructions of the' manufacturer. In order to test the expression, 106 Sf9 cells were infected with recombinant Baculovirus and an m.o.i. 15 (multiplicity of infection) of 5 to 10. After the incubation, the medium was removed and the cells washed with PBS (140 mM NaCl, 2.7 mM Kcl [sic], 8.1 mM Na 2

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4 , 1.5 mM KH 2

PO

4 , pH 7.2). The cells were then lysed in SDS sample buffer and tested by SDS gel chromatography and 20 immunoblot assay. 3. Purification of virus-like particles For the preparation of CVLPs, Trichoplusia ni (TN) High 25 Five cells were cultured at 27 0 C up to a density of 1-1.5 x 106 cells per ml in Ex-Cell 405 serum-free medium (JRH, Biosciences, Lennexa [sic], KS). A 400 ml culture was harvested and infected with an m.o.i. of 2 to 5 with recombinant baculoviruses for one hour with 30 periodic inversions. Up to 240 ml of medium were added and the cells grew for 3 to 4 days. The cells were then pelleted and resuspended in 10 ml of extraction buffer (10 mM MgCl 2 , 1-50 mM CaCl 2 , 150 mM NaCl, 20 mM Hepes, 0.01% Triton (optional), pH 7.4) and sonicated for 35 45 seconds at 60 watts. After centrifugation at 10,000 rpm in a Sorvall SS34 rotor, the pellet was dissolved in 6 ml of extraction buffer, sonicated for 30 seconds at 60 watts and centrifuged again. The WO 99/48518 - 19 - PCT/EP99/01996 supernatants were combined and applied to a two-stage gradient of 40% (w/v) succhrose [sic] and 57.5% (w/v) CsCl. After centrifugation in an SW-28 rotor at 27,000 rpm for two hours, the interphase and the CsCl 5 layer were collected, adjusted to a CsCl density of 1.38 g/ml and centrifuged at 45,000 rpm for 16 hours. The gradients were fractionated and each fraction was tested by Western Plot [sic] using anti-HPV16LlmAb Camvirl (Pharningen [sic], San Diego, CA). The reactive 10 fractions were combined and dialysed by means of an ultrafiltration using a Centricon 30 microconcentrator (Amicon Corp. Beverly, MA) against Hepes buffer (1 mM Hepes, 149 mM NaCl, 0.5 mM KCl, pH 7.2) and the presence of CVLPs was confirmed by means of 15 transmission electron microscopy. The concentration of LlE7 protein was determined approximately, in an SDS gel which was stained with Coomassie blue, by comparison with BSA standards. 20 4. Culturing of mouse cells The C57BL/6-derived mouse cells TC-1 are primary pulmonary epithelial cells of C57BL/6 mice, which have been transformed by transfection with the oncogenes 25 HPV-16 E6 and E7 and c-Ha-ras (Lin, K.-Y. (1996), supra). RMA (Ljungrenn & Karre (1985) J. Exp. Med., 162, 1745-1757) and the processing-defective cell line RMA-S are C57BL/6 thymoma cells. The HPV-16 E7-transfected RMA-E7 is described in Speidel, K. 30 et al. (1997) Eur. J. Immunol., 27(9), 2391-2399. All cells were cultured in RPMI-1640, supplemented with 10% FCS, 2-ME, L-glutamine and antibiotics. 0.8 mg/ml of G418 (Gibco BRL, Gaithersburg, MD) was added for the culturing of RMA-E7 transfectants. TC-1 cells 35 additionally grew in the presence of 0.4 mg/ml of G418, 0.2 mg/ml of hygromycin and 1 mM sodium pyruvate.

WO 99/48518 - 20 - PCT/EP99/01996 5. Production of cytotoxic T-cell (CTL) lines 10-14 days after immunization, the spleen cells were prepared; 2-4 X 107 spleen cells were cocultured with 5 10s irradiated (200 Gy) syngenic RMA-E7 transfectants per ml. After 5 days, the first 51 Cr-release cytotoxicity test was carried out. For the production of CTL lines, the spleen cells were restimulated weekly in 24-hole plates with 2 x 105 irradiated RMA-E7 10 transfectants per hole as stimulator cells and 5 x 106 irradiated (33 Gy) C57BL/6 spleen cells per hole as feeder cells. 6. 5 Cr-release cytotoxicity test 15 For the 5'Cr-release cytotoxicity test, effector T cells were added to 1 x 104 5'Cr-labelled target cells per hole on a 96-hole plate having a different effector cell to target cell ratio (E:T) . The target cells were 20 labelled with Na 2 51 CrO 4 (100 yCi per 2 X 106 cells) for one hour at 37 0 C. The peptide having the sequence RAHYNIVTF (amino acid 49-57 of HPV-16 E7; Feltkamp, M.C.W. et al. (1993), Eur. J. Immunol., 23, 2242-2249) was added during this incubation in a concentration of 25 50 pM. In the peptide titration test with a constant E:T ratio, the 51Cr-labelled target cells were cultured with decreasing peptide concentrations (1 gg - 100 pg per ml) for one hour at 37 0 C before the effector cells were added. After incubation at 37 0 C for four hours, 30 50 yl of the supernatant per hole were transferred to a Luma plate (Packard) and dried. The radioactivity was measured in a $-counter (Trilux Microbeta, Wallac). The mean specific lysis was calculated according to the following formula: % specific lysis = (experimental 35 release - spontaneous release)/(complete release -- spontaneous release) x 100.

WO 99/48518 - 21 - PCT/EP99/01996 7. Induction of E7-specific cytotoxic T lymphocytes by CVLPs 11 six to sixteen week-old female C57BL/6 mice were 5 immunized with 1-20 jig of CVLPs without adjuvant by means of a simple s.c. injection. Two weeks later, the spleen cells were prepared and stimulated in vitro with HPV-16 E7-expressing transfectants of the C57BL/6 derived tumour cell line RMA (RMA-E7) as stimulator 10 cells. After culturing for five days, the first cytotoxicity test was carried out. The stimulation of the spleen cells was repeated at weekly intervals. As shown in Fig. 1, immunization with 15 1 Mg of LlACE7 1 -6 0 CVLPs, i.e. the carboxy-terminal 34 amino acids of HPV-16 Ll were replaced by the amino acids 1-60 of HPV-16 E7, led to a strong E7-specific CTL response. 20 Altogether, after five days a specific lysis of RMA-E7 transfectants of between 7.7% and 54.7% (average value 33.6%; E:T ratio 8:1-33:1) was observed. The average lysis after the second stimulation was between 35.8% and 57.8% (average value 45.3%; E:T ratio 8:1-44:1). If 25 the mice were only immunized with 1 gg of LlACE7 1

-

60 CVLPs, on average, after a culturing time of 5 days, a lower reactivity of E7-specific CTLs was observed (7.7-49.5%; average value 28.5%) than after immunization with 5 gg (17.2-44.7%; average value 30 33.9%) or 20 Mg (26.0-54.7%; average value 38.5). The spleen cells of the control mice (vaccinated with HBS buffer) showed no E7-specific reactivity after five days (0.3 - 8.9%, average value 3.4%, E:T ratio 4:1-9:1). 35 L1ACE7 1 -6 0 CVLP contains a known H2-Db T-cell epitope of the E7 protein (E7 4 9

_

5 7 , Feltkamp, M.C.W. et al. (1993), \ supra). In order to confirm the obtained E7 specificity WO 99/48518 - 22 - PCT/EP99/01996 of the CTLs, they were therefore tested against RMA cells which are loaded with the peptide E7 4 9

-

5 7 . CTLs which recognize RMA-E7 transfectants showed a specific cytotoxic activity against E7 4 9

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57 RMA or RMA-S target 5 cells. The measured specific lysis varied between 21.2-25% after five days (E:T ratio 8:1-15:1, see Fig. 1). In the peptide titration test carried out, after the third stimulation RMA-S cells were lysed to 70.5% or 71.4% (two CR lines were tested) after loading 10 with E7 4 9 -57 at a concentration of 1 pg/ml (E:T ratio 40:1, see Fig. 2). Even if RMA-S cells were provided with as little as 100 pg per ml of E7 4 9

_

57 peptide, a specific lysis of 36.2% and 47.2% was obtained. CTLs which are loaded with a control peptide, i.e. the 15 influenza virus nucleoprotein-derived peptide having the amino acids 366-374, which represent a Db-CTL epitope, were not recognized. Spleen cells which were isolated from C57BL/6 mice after treatment with L1ACE7 1

-

5 5 CVLPs (E7 sequence truncated by five amino 20 acids), did not recognize RMA-E7 cells, even if the mice were vaccinated with an increasing dose of up to 250 yg. From these data, it is concluded that the CTLs were 25 induced by L1ACE7 1

-

6 0 CVLPs, are E7-specific and recognize the peptide E7 4 9

-

5 7 with high affinity. In an FACS analysis of representative CTL lines, the CTLs were identified as CD8-positive. 30 8. Prevention of the growth of a syngenic E7-expressing tumour in C57BL/6 mice In order to determine whether the vaccination with L1ACE7 1

-

6 0 CVLPs induce [sic] an effective immunity 35 against syngeneic tumour cells (TC-1, Lin, K-Y. (1996), Cancer Res. 56, 21-26), tumour protection experiments were carried out. C57BL/6 mice (3-5 per group) received 1 s.c. injection of 10 Mg of L1ACE7 1 -6 0 CVLPs, 10 yg of WO 99/48518 - 23 - PCT/EP99/01996 L1ACE7 1

-

5 5 CVLPs, 10 gg of L1AC VLPs or an equivalent volume of HBS buffer without adjuvant. Two weeks after the immunization, 6 x 104 TC-1 cells, suspended in 200 yl of PBS, were inoculated into the left flank of 5 the mice. The tumour size was measured twice per week for 2 months until the mice were killed. The results of all experiments are summarized in Table 1. The total tumour growth rate in the mice which 10 were vaccinated with HBS buffer, L1AC VLPs or L1ACE7 1

-

55 CVLPs was on average 80.5%. Vaccination with L1AC CVLPs did not protect against the tumour, although delayed tumour growth was observed (see Fig. 3). However, vaccination of mice with L1ACE7 1 -6 0 CVLPs protected 15 against tumour growth: only one mouse (1/13) developed a small, slowly growing tumour after 38 days, which regressed within a two-month period. Spleen cells of HBS or L1AC VLP-vaccinated mice which 20 developed TC-1 tumours showed no or only small levels of an E7-specific CTL response, independently of whether the mice had developed tumours or not. After the 2nd stimulation, an E7-specific lysis of RMA-E7 target cells of between 0-21% in tumour-bearing mice 25 (average value 14%, E:T ratio 5:1-24:1), and of between 0 and 22.2% (average value 10.4%, E:T ratio 7:1-40:1), was observed in tumour-free mice. In contrast to this, in mice which were protected against tumour growth after the immunization with CVLP L1ACE7 1

-

6 0 , an E7 30 specific CTL response of between 29.1 and 49.8% (average value 36.9%, E:T ratio 11:1-24:1) was found after the 2nd stimulation. It was furthermore analysed whether the vaccination 35 leads to a regression of already existing tumours. Two weeks after the inoculation of 6 x 104 TC-1 cells (s.c.), the mice (5 per group) received a single ) injection of 10 gg of LlACE7 1

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6 0 CVLPs, 10 gg of WO 99/48518 - 24 - PCT/EP99/01996 VLP LlAC or HBS buffer without adjuvant. In this experiment (see Fig. 4), the tumour development of control mice which were either treated with HBS or immunized with VLP LlAC was only 60% (3 of each group, 5 6/10). However, all mice (5/5) which were vaccinated with LlACE7 1

-

60 CVLPs generated an immune response against the established tumours and remained tumour free two months after the tumour cell injection. O ~\:

Claims (18)

1. Medicament for the avoidance or treatment of human papillomavirus (HPV)-specific tumour comprising at 5 least one fusion protein from at least one Ll protein of one or more papillomaviruses and at least one E protein of one or more papilloma viruses and, if appropriate, suitable additives and/or excipients, characterized in that the 10 fusion protein contains no papillomavirus unspecific epitopes.

2. Medicament according to Claim 1, characterized in that the tumour is a carcinoma of the larynx, 15 cervix, penis, vulva or anus.

3. Medicament according to Claim 1 or 2, characterized in that the medicament contains no adjuvant. 20

4. Medicament according to one of Claims 1-3, characterized in that the additive or excipient is about 0.3 to about 4 M, preferably about 0.4 to about 3 M, in particular about 0.5 to 2 M, 25 especially about 1 to about 2 M, of a salt having a pH of about 7.3 to about 7.45, preferably about

7.4. 5. Medicament according to Claim 4, characterized in 30 that the salt is an alkali metal or alkaline earth metal salt, preferably a halide or phosphate, in particular an alkali metal halide, especially NaCl and/or KCl. 35 6. Medicament according to Claim 4 or 5, characterized in that the pH is adjusted using a buffer, preferably using a phosphate buffer, tris buffer, HEPES buffer or MOPS buffer. WO 99/48518 - 26 - PCT/EP99/01996 7. Medicament according to one of Claims 1-6, characterized in that the L protein is a deleted L protein, preferably a deleted Li and/or 5 L2 protein.

8. Medicament according to Claim 7, characterized in that the L protein is a C-terminally deleted L protein, in particular a C-terminally deleted 10 L1 protein.

9. Medicament according to Claim 7 or 8, characterized in that up to about 35 amino acids are deleted from the L protein, preferably about 15 25 to about 35 amino acids, in particular about 32 - 34 amino acids.

10. Medicament according to one of Claims 1-9, characterized in that the E protein is a deleted 20 E protein, especially a deleted E6 and/or E7 protein.

11. Medicament according to Claim 10, characterized in that the deleted E protein is a C-terminally 25 deleted E protein, preferably a C-terminally deleted E7 protein.

12. Medicament according to Claim 10 or 11, characterized in that up to about 55 amino acids 30 are deleted, preferably about 5 to about 55 amino acids, in particular about 38 to about 43 amino acids.

13. Medicament according to one of Claims 1-12, 35 characterized in that the fusion protein is present in the form of a capsid and/or capsomer. WO 99/48518 - 27 - PCT/EP99/01996

14. Medicament according to one of Claims 1-13, characterized in that the HPV is selected from HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-42, HPV-45, HPV-52 and/or 5 HPV-58.

15. Medicament according to Claim 14 in the form of a combination vaccine, characterized in that the papillomaviruses are selected from HPV-16 and 10 HPV-18 or HPV-18, HPV-31, HPV-45 and HPV-58 or HPV-6 and HPV-11.

16. Medicament according to one of Claims 1-15, characterized in that the fusion protein is a 15 L1ACE7 1 _ fusion protein, preferably in the form of a CVLP, in particular of HPV-16, x being an integer from 55 up to and including 60.

17. Medicament according to Claim 16, characterized in 20 that the fusion protein is an L1ACE7 1 - 5 5 or L1ACE7 1 -6 0 fusion protein.

18. Process for the production of a medicament according to one of Claims 1-17, characterized in 25 that a cell comprising an expression vector which codes for the fusion protein mentioned is cultured under suitable conditions, the expression product is isolated and, if appropriate, suitable additives and excipients are added. 30

19. Process according to Claim 18, characterized in that the expression vector contains no nucleic acid sequences which extend the fusion protein mentioned by further amino acids during 35 expression.

20. Use of a fusion protein according to one of Claims 1 and 7-17 for the production of a WO 99/48518 - 28 - PCT/EP99/01996 medicament, on the one hand for the prevention of HPV-specific tumours and on the other hand for regression of already existing HPV-specific tumours.